The measurement of various body constituents by the use of radioimmunoassay techniques has achieved widespread acceptance in recent years. Exemplary of substances which can be measured by radioimmunoassay using currently available commercial kits are ACTH (adrenocorticotropin), aldosterone, angiotensin I, angiotensin II, barbiturates, cyclic AMP, cyclic GMP, digoxin, folic acid, FSH (follicle stimulating hormone), gastrin, HB.sub.s Ag (hepatitis B antigen), HGH (human growth hormone), insulin, TSH (thyroid stimulating hormone), T4 (thyroxine), T3 (triiodothyronine), and vitamin B12.
Yalow and Berson, In Vitro Procedures With Radioisotopes In Medicine, International Atomic Energy Agency, Vienna (1970) pgs. 455 et seq., express the principle of radioimmunoassay in the following terms:
"Unlabelled antigen in unknown samples competes against labelled antigen ("tracer") for binding to antibody and thereby diminishes the binding of labelled antigen. The degree of competitive inhibition observed in unknown samples is compared with that obtained in known standard solutions for determination of concentration of antigen in unknowns."
The above-described type of radioimmunoassay procedure has come to be known as the "indirect" method of radioimmunoassay. Alternatively, the "direct" method of radioimmunoassay can be used to determine the presence or absence of a particular antigen in an unknown sample. In the "direct" method, labelled antibody is mixed with the unknown sample, which if it contains the antigen in question, will bind the labelled antibody. One particular type of "direct" radioimmunoassay, known as the "sandwich" technique, may be used for the determination of the presence of antigens which have at least two antigenic sites. The "sandwich" technique comprises adding a sample (which may or may not contain antigen) and labelled antibody to unlabelled antibody. If the sample contains antigen it will bind to the unlabelled antibody and will in turn provide a binding site for the labelled antibody.
In all radioimmunoassay procedures it is necessary to provide means for separating the bound from the free labelled tracer material. Many widely varied procedures have been developed and used; exemplary procedures are electrophoresis; chromatography; ion exchange; adsorption to dextran-coated charcoal, talc, or cellulose; and a number of solid-phase antibody techniques.
Two of the widely recognized solid-phase separation techniques comprise the covalent chemical bonding of an antibody to an insoluble polymeric substance or the physical adsorption of an antibody onto an insoluble polymeric substance; see, for example, Gurvich et al., Nature, 203:648 (1964); Wide et al., Biochim. Biophys. Acta., 130:257 (1966); Catt et al., Biochem. J., 100:31c (1966); Catt et al., J. Lab. Clin. Med., 70:820 (1967); Catt et al., Nature, 213:285 (1967); Axen et al., Nature, 214:1302 (1967); Catt et al., Science, 158:1570 (1967); Wide et al., Lancet, 2:1105 (1967); Salmon et al., J. Immunol., 103 (1):129 (1969); Catt, U.S. Pat. No. 3,646,346, issued Feb. 29, 1972; and Axen et al., U.S. Pat. No. 3,645,852, issued Feb. 29, 1972. The principal advantage of the solid-phase antibody separation techniques in radioimmunoassays is that they allow the isolation of bound from free labelled tracer material to be carried out by a simple washing step at the completion of the immune reaction. This washing step may in practice, however, require several manipulations by the laboratory technician.
U.S. Pat. No. 3,645,852 teaches that cyanogen halides can be used to covalently link water-soluble proteins and water-soluble peptides containing a primary or secondary amino group to a water-insoluble polymer such as cellulose. Catt et al., J. Lab. Clin. Med., 70:820 (1967), discuss the use of a polymeric disc (polytetrafluoroethylene-g-isothiocyanatostyrene) coated with antibody, instead of antibody coated polymer powder, as separation means in a radioimmunoassay.
The apparatus in which radioimmunoassay test procedures are performed can be a critical factor in determining the accuracy and reproducibility of the tests. Catt, in U.S. Pat. No. 3,646,346, issued Feb. 29, 1972, combined into a single entity the apparatus in which the immunochemical reaction is run and the means for separating bound from free labelled tracer material. The solid phase system of Catt, known as the "anti-body-coated tube" system comprises coating the interior of a water-insoluble polymeric test tube with antibodies against the protein to be determined; adding to the test tube an aqeuous sample containing the protein; adding to the test tube the same protein labelled with a radioisotope; aspirating the liquid from the test tube and washing the test tube; and measuring the radiation emitted by the liquid or by the test tube.
Beall et al., in U.S. Pat. No. 3,932,141, issued Jan. 13, 1976, disclose an apparatus useful in the performance of multiple radioimmunoassay test procedures. The apparatus comprises a receptacle tray with wells; polymeric balls coated with antibody to be placed in the wells; and a holder with release mechanism to be used in depositing and removing the balls from the wells. The radioimmunoassay procedure described by Beall et al. includes the use of aspiration to remove liquids from the wells.